Synthesis and characterization of a macrocyclic near-infrared optical scaffold

Article abstract of DOI:10.1021/ja034186o

Macrocyclization of a bioactive peptide with a bifunctional near-infrared fluorescent optical probe gives a compound that retained the receptor binding affinity of the peptide and the photophysical properties of the optical probe. The robust nature of the new compound provides a structural framework for optimizing the activity of bioactive molecules and for monitoring chemical or biological processes in vivo and in vitro by near-infrared optical methods. Copyright

Full text of DOI:10.1021/ja034186o

Published on Web 06/05/2003
Synthesis and Characterization of a Macrocyclic Near-Infrared Optical
Scaffold
Yunpeng Ye,† Wen Ping Li,† Carolyn J. Anderson,† Jeffery Kao,‡ Gregory V. Nikiforovich,§ and
Samuel Achilefu*,†
Departments of Radiology, Chemistry, and Biochemistry and Molecular Biophysics, Washington UniVersity,
St. Louis, Missouri 63110
Received January 15, 2003; E-mail: achilefus@mir.wustl.edu
Scheme 1. Synthesis of NIR Macrocyclic Optical Scaffolda
Fluorescence-emitting molecules continue to play a major role
in all facets of chemistry, materials science, and life sciences. With
the advent of molecular medicine, the challenge for chemists is to
develop new photoactive molecules that can probe specific molec-
ular events and inhibit pathophysiological processes. Light in the
near-infrared (NIR) wavelengths is particularly attractive to study
chemical and biological processes in tissue because its absorption
and scattering by endogenous biomolecules are minimal and, hence,
can penetrate into deep tissues without the accompanying autof-
luorescence that occurs in the UV/visible wavelengths.1,2
Recent studies have shown that conjugating NIR fluorescent
a (i) Ac2O/TEA, (ii) BrC2H4CO2H, (iii) CH3COONa, (iv) (a) PyBOP,
(b) TFA. Compound 6′ is the protected peptide on resin.
compounds to bioactive peptides and proteins is a reliable method
to selectively deliver the optical probes to tumors, resulting in the
localization and treatment of the target pathologic tissue.3-5 For
small peptides, the fluorophore is typically attached to the N-
terminus of the bioactive carrier. Unlike proteins, small peptides
are highly susceptible to rapid degradation by proteolytic enzymes.
One approach to improve metabolic stability of small peptides and
to rigidify their low-energy bioactive conformations is to use head-
to-tail cyclic peptide scaffolds.6,7
Accordingly, we report the first NIR macrocyclic fluorescent
bioactive peptide scaffold that has great potential for a variety of
applications. In a model system, we demonstrate that macrocycli-
zation of a somatostatin receptor-avid nanopeptide with a bifunc-
tional NIR carbocyanine optical probe gives a compound that re-
tained the high receptor binding affinity of the peptide and the ab-
sorption and fluorescence emission properties of the fluorescent
probe. NMR and computational modeling data show that the
macrocyclization did not significantly perturb the established low-
energy conformation of the peptide.
protected lysine. The C-terminal lysine residue provides the second
amino group for intramolecular cyclization on solid support. The
choice of 8 has a practical implication because the sst receptor is
up-regulated in a variety of human tumors.9 Thus, starting with a
preloaded Fmoc-Lys(Dde)-OH on Rink amide resin, the peptide
was synthesized on a solid support by a standard procedure. While
the peptide was still on the solid support, simultaneous removal of
the orthogonal Dde and Fmoc protecting groups of R-dF1 and ꢀ-K9
amino groups and subsequent reaction with the PyBOP preactivated
carboxyl groups of 5 gave the desired macrocyclic compound (7)
after cleavage from the resin with TFA (Scheme 1). The spectral
properties of 7 (λmax,abs 792; λmax,em 811 nm) were similar to those
of 5 (λmax,abs 786; λmax,em 811 nm) in 25% aqueous DMSO.
Expectedly, the molar absorptivity of the dye (5) decreased from
2.97 × 105 to 1.1 × 105 cm-1 M-1 in 7 due to a combination of
macrocyclization and “dye dilution” effects resulting from the
addition of the peptide moiety.
A major shortcoming to using NIR carbocyanine fluorescent
probes in research is the difficulty in synthesizing high purity
reactive intermediates in high yields by conventional methods. A
review of the literature8 shows that N-acetylation of glutaconalde-
hyde is an important step in the synthesis of NIR dyes. This acet-
ylation is traditionally performed in situ and purification of the final
product is very demanding. We observed that pre-acetylating gluta-
conaldehyde (1) prior to reaction with activated benzoindole (4)
gives a good yield (>60%) of the desired bifunctional NIR dye
(cypate, 5) without the need for HPLC purification (Scheme 1).
To prepare the macrocyclic molecule from 5, lysine was incor-
porated at the C-terminus of a cyclic somatostatin (sst) receptor-
avid octapeptide [dF-cyclo(C2-Y3-dW4-K5-T6-C7)-T8; octreotate;
8]. While it is possible to use the ꢀ-amino group of K5 for the
cyclization, this amino acid is part of the octapeptide’s pharma-
cophore and, hence, we chose to introduce a second amino group
at the C-terminus by using the readily available orthogonally
To evaluate the effect of incorporating lysine at the C-terminus
of 8 and subsequent macrocyclization of the peptide, we performed
the receptor-binding assays of these compounds. The IC50 values
of 8, K9-octreotate [dF1-cyclo(C2-Y3-dW4-K5-T6-C7)-T8-K9-
NH2; 6], and the macrocyclic analogue [cyclo cypate-(dF1-cyclo-
(C2-Y3-dW4-K5-T6-C7)-T8-K9)-NH2; 7], which were obtained by
competition with 111In-DTPA-octreotide, were 0.12, 21.68, and 8.17
nM, respectively. This result shows that macrocyclization retained
the receptor binding affinity of the precursor peptide 6 and that
the IC50 value of 7 is comparable to those of previously reported
linear analogues.5 A preliminary stability study by HPLC of 7 and
the linear analogue (cypate-octreotate)5 in serum after a 24 h
incubation at 37 °C shows a decrease of <10% of the macrocyclic
compound and >25% of the linear analogue. This result suggests
that macrocyclization enhanced the stability of the new construct.
Assuming that the bound conformation is selected by a receptor
from the set of low-energy conformers of the peptide in solution,
one can conclude that peptide 7 retained receptor binding affinity
because its low-energy conformations are not significantly affected
by macrocyclization. This conclusion was confirmed by NMR and
† Department of Radiology.
‡ Department of Chemistry.
§ Department of Biochemistry and Molecular Biophysics.
9
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10.1021/ja034186o CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
between nitrogen atoms of 12.3 Å (according to model building
employing the readily available SYBYL computational package).
Consequently, we do not expect that all low-energy conformers of
9 are compatible with attachment of the dye, but only those pos-
sessing the suitable distance between the R-amino group of dF1
and the ꢀ-amino group of K9. Our calculations did not consider
multiple low-energy conformations for the side chain of K9, but it
is quite reasonable to expect that the option selected by the optimi-
zation procedure11 and energy minimization is, at least, one of the
lowest energy options possessing the highest statistical weight in
solution. Therefore, selection of low-energy conformations with the
RN1-ꢀN9 distance in the range of 12 ( 2 Å will rather accurately
define the conformers of 9 most suitable for attachment of the dye.
Such a selection yielded 66 conformations satisfying the above
requirement. This new limited ensemble retains most of the features
of the extended one: it has almost the same percentage of the â-
reversal-like structures (ca. 77%), and again, all close interproton
distances observed in the NMR experiments can be reproduced by
this limited ensemble as a whole. This is in agreement with experi-
mental finding that the same close interproton contacts are charac-
teristic of both 8 and compounds with the dye attached (7). Several
conformers possessing the most (but not all) individual interproton
distances satisfying the NMR data belong to the same cluster of
geometrically similar structures (rms < 2 Å, CR atoms only). Figure
1b shows a representative structure from that cluster.
One advantage of the macrocyclization approach is the possibility
of constraining the bioactive conformations of linear peptides that
may adopt a more flexible conformation. For example, bioactive
linear bombesin peptide analogues that possess a random coil struc-
ture,12 may be forced to adopt a partial â-sheet or helical confor-
mation after cyclization. The robust nature of the structural frame-
work of these macrocyclic compounds lends itself to a variety of
applications, including optimizing and monitoring by NIR optical
methods the bioactivity of putative drugs, improving the in vivo
metabolic stability of fluorescent drugs, enhancing receptor recogni-
tion, modifying spectral properties by altering the macrocyclic ring
size, and elucidating the low-energy conformations of small linear
biomolecules that would otherwise be difficult to obtain.
Figure 1. (a) Expansion of NH-RH region of 600 MHz NOESY spectra
and (b) sketch of a representative structure of 7.
computational modeling data. Proton chemical shifts of 7 were
compared with those of 6 and 8. The proton chemical shifts were
assigned by analysis of TOCSY and NOESY spectra. Sequential
assignments were obtained by using the NH-RH fingerprint region
of the NOESY spectra. All the dRN connections between adjacent
residues were observed and a continuous path indicates the segment
from dF1 to T8 or K9 C-terminal residue. Cyclization through
disulfide S-S bond was evidenced by several observed NH-RH
NOEs between dF1 and C2 as well as between C7 and T8. Strong
NOE between dF1NH and cypate CH2, and between K9ꢀNH and
cypate CH2 and cypate-aromatic protons, confirm the dF1-cypate-
K9 linkage in compound 7. The presence of several RΗi-NHi+2
contacts such as C7-K5, T6-W4, and K5-Y3 suggests a tight
â-reversal conformation of the 20-membered cyclic disulfide
peptide. Furthermore, the observation of RH-RH NOE for the
residues from C2 to C7 indicates the backbone at the cyclic moiety
may deviate from the trans-amide conformation. Of particular
interest is the observation of T6NH-W4RH, T6NH-Y3RH, T6NH-
C2RH, and T8NH-T6RH NOE cross-peaks, suggesting the disulfide
ring closure causes a â-reversal of the peptide in vicinity of T6
bringing T8, W4,Y3, and C2 in close proximity. All three peptides
have similar sequential and RΗi-NHi+2 NOEs except that the T8NH-
T6RH and C7NH-K5RH NOEs were not observed in compound 7
(Figure 1a). This indicates that an additional K9 residue and the
cypate linkage may cause the 20-membered ring to adopt a less
compact topology with a conformational change appearing only at
the residues near the C-terminus of 7.
Acknowledgment. We thank the NSF (BES-0119489, S.A.),
NIH (GM53630, G.V.N.; CA64475, C.J.A.), and Siteman Cancer
Center Barnard Research Fund (S.A.) for financial support and G.
R. Marshall and W. J. Welch for advice.
Supporting Information Available: Detailed descriptions of
synthetic, NMR, and computational modeling procedures. This material
is available free of charge via the Internet at http://pubs.acs.org.
Since the NMR data did not show a strong NOE between the
peptide and the fluorescent probe, the computational modeling was
restricted to the key common element for compounds 6 and 7,
namely, Ac-dF1-cyclo(C2-Y3-dW4-K5-T6-C7)-T8-K9-NH2 (9).
Energy calculations were performed in general accordance with a
published procedure10 (see Supporting Information for detailed des-
cription). The most characteristic feature of the obtained ensemble
of low-energy conformers (206 structures of peptide backbone) is
the distinct â-reversal centered on the dW4-K5 fragment. About
79% of low-energy structures possess that feature as compared to
the six-residue â-reversal template (rms less than 2 Å, CR atoms
only). None of the individual structures satisfied all the 13 nonse-
quential interproton restraints suggested by NMR measurements.
However, the ensemble of the found low-energy structures as a
whole fully satisfies the NMR restraints, since for every experi-
mentally observed NOE it is possible to find the corresponding
interproton distance in the range under 4.5 Å in a number of low-
energy conformers, which comprise from 6% to 52% of the entire
ensemble depending on the particular NOE.
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The dye itself is conformationally rigid, since its polyethylene
chain most likely is fixed in all-trans configuration with the distance
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